Investigation of biocidal efficacy of commercial disinfectants used in public, private and workplaces during the pandemic event of SARS-CoV-2

This study investigated the performance of 24 commercial disinfectants present on the market during last year according to the manufacturer’s instructions. Recently, national and international organizations of public health performed studies on disinfection products due to the increasing awareness of the potential and growing risks on human health, such as skin damage and reactions in the mucosal lining, especially for the healthcare workers in their frequent daily use. However, there are many limitations in the common cleaning/disinfection products on market as in the selection of effective disinfectants to decontaminate inanimate surfaces. We analyzed the disinfection power of hydrogen peroxide, quaternary ammonium compounds, alcohols, phenols and aldehydes used as active principles according to international guidelines. The antimicrobial properties were assessed by broth microdilution, and antibiofilm properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus); their virucidal efficacy was tested against Herpes simplex virus type 1 (HSV-1) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The quaternary ammonium compounds demonstrated better efficacy than others and in some cases ready to use products had also virucidal and antimicrobial activities after dilution at 0.125%. The scientific evidence indicates that many commercial products are used at high concentrations and high doses and this could have deleterious effects both on human health and the environment. A lower concentration of active ingredients would avoid the excessive release of chemicals into the environment and improve skin tolerance, ensuring the health and safety protection of workers, including the healthcare operators at their workplace.

Bacteria/viral strains and cell culture conditions. The bacterial strains used to assess the antibacterial activity and the biofilm degradation were E. coli and S. aureus, as representative of Gram-negative and Grampositive, respectively. All strains were purchased from the American Type Culture Collection (ATCC) (Manassas, USA).In detail, for the antibacterial assays E. coli ATCC 11229 and S. aureus ATCC 6538 were used; for the biofilm degradation assays E. coli ATCC 25992 and S. aureus 25923 were chosen as biofilm producers, while S. aureus ATCC 6538 and E. coli ATCC 11229 as non-biofilm producers. HSV-1 (strain SC16) and SARS-CoV-2 (strain VR PV10734, kindly donated by the Lazzaro Spallanzani Hospital of Rome, Italy) were propagated on Vero cell line (ATCC CCL81). Eagle's Minimum Essential Medium (EMEM) supplemented with 2 mM L-Glutamine, 100 IU/mL penicillin-streptomycin solution, and 10% Fetal Bovine Serum (FBS) were used for the cell growth.

Determination of minimum inhibitory concentration (MIC).
The antimicrobial activity was conducted following the broth microdilution method, following the guidelines of the National Committee on Clinical Laboratory Standards (NCCLS). In detail, E. coli and S. aureus were plated on Brain Heart Infusion (BHI) agar plates (Sigma-Aldrich, Missouri, USA) and incubated at 37 °C overnight (O. N.). A fresh colony of both bacteria was inoculated in BHI-broth (Sigma-Aldrich, Missouri, USA) and incubated at 37 °C under vigorous orbital shaking (180 rpm) for 20 h. The following day, 300 µL of bacterial suspension was inoculated in fresh BHI-broth and incubated at 37 °C at 180 rpm until mid log-phase growth (6 × 10 8 colony-forming units (CFU/ mL). Three serial dilutions were performed to obtain a final bacterial concentration of 1 × 10 6 CFU/mL. Fifty microliters of bacterial inoculum were added to each well of a sterile 96-well plate. Meanwhile, the 24 com-

Data analysis.
All the experiments were performed in independent triplicate and results were expressed as mean ± standard deviation. Statistical analysis was performed by using ANOVA: P-value, R-square and F value were calculated to determine the significance of the results. Statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). The results were considered significant at P-value < 0.05 and R-square > 0.8. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Results
Disinfectant antibacterial activity. The antibacterial activity of 24 currently commercially available compounds was tested against E. coli and S. aureus (Table 2) Fig. 3-4).
Disinfectant antiviral activity. The antiviral activity of 24 disinfectants was explored against DNA and RNA enveloped viruses (HSV-1 and SARS CoV-2, respectively). We tried to mimic a real condition in which a surface disinfectant could be used. In detail, we tested the virucidal activity of the disinfectants on a contaminated surface. Then, to quantify the antiviral power of each compound avoiding its normal cytotoxicity, we infected a cell monolayer, as described in "Biofilm degradation assay", as soon as the mixture of virus and compounds evaporated.. Basically, the virus was left to interact on a plastic surface with a single drop of each compound at the concentrations reported in Table 4 Then, the mixture was left to evaporate for 1 h. Subsequently, each mixture was diluted to infect the cell monolayer at 0.01 MOI, which was incubated for the time of virus adsorption (1 h for HSV-1 and 2 h for SARS-CoV-2). As shown in Supplementary Materials Fig. 5, the compounds, D 1-2-8-12-13-14-15-16-18-20-21-22-24 were not active against both viruses, while the other disinfectants showed remarkable dose-dependent inhibition of the viral replication. Data showed that most compounds exhibited similar inhibitory activity against HSV-1 and SARS-CoV-2 at the same concentrations. In detail, setting IC90 as the treshold line, D3-5-6-9-10-11-17-19-23 exhibited inhibitory activity with IC90 at 0.025, 12.5, 0.078, 0.043, 0.008, 0.037, 0.008, 0.093, and 210 g/L, respectively. Only two of 24 disinfectants (D4 and D7) showed a different efficacy of inhibition against DNA and RNA viruses. Indeed, D4 interfered with HSV-1 infection with 90% inhibition at 0.075 g/L, and SARS-CoV-2 at 0.037 g/L; while D7 showed better inhibitory activity against SARS-CoV-2 with IC90 at 0.375 g/L compared to HSV-1 with IC90 at 3 g/L.

Discussion
Since last year, due to the pandemic of coronavirus disease, the use of disinfectants to prevent microbial infections is rapidly increasing worldwide [20][21][22][23][24][25][26] . Numerous studies analyzed the role of safe for humans chemical agents with high bactericidal and virucidal potential [27][28][29][30][31] . However, currently there is still very little knowledge about the specific disinfectant agents for this new virus with a biosafety level-3 (BSL-3). For this reason, international organizations recommended active agents tested against the other non potentially dangerous coronaviruses. However, it's known that even viruses within the same family could respond differently to a given disinfectant and scientific results disagree about which disinfectants would be more efficaceous against SARS-CoV-2. In our study, we analysed the efficacy of some commercial products available on market to sanitize surfaces and to understand the real efficaceous concentration in use both for antibacterial and virucidal capacity. Quaternary ammonium compounds (QACs) based disinfectants play an important role in veterinary medicine and the control of animal diseases [32][33][34] . QACs are cationic surface active detergents widely used for the control of microorganisms in clinical and industrial environments. QACs are commonly used as antimicrobial pesticides, even if their biocidal properties were under the spotlight in recent years highlighting also the disadvantages 35,36 . The QACs disadvantages were associated with the presence of long alkyl chains that can induce fatal damage to a wide variety of organisms; they can bind to negatively charged lipid bilayers and lead to dissociation of the cell membrane components and leakage of intracellular components, resulting in cell death 37,38 . In particular, databases for Didecyl dimethyl ammonium chloride (DDAC) and alkyl (C12, C14, C16) dimethyl benzyl ammonium chloride (C12-C16 ADBAC) are complete to support the registered uses of these pesticides. DDAC and C12-C16 ADBAC are permanently charged cationic compounds, and available studies indicate that both DDAC and C12-C16 ADBAC are poorly absorbed via the oral and dermal exposure routes, and are primarily eliminated in feces. ECHA 39 and EPA 40 concluded that oral and dermal absorption of DDAC and C12-C16 ADBAC does not exceed 10%. Low dermal and oral absorption of DDAC and C12-C16 ADBAC is consistent with the lack of systemic toxicity observed across available repeated doses from oral and dermal toxicology studies conducted with beagles, mice, and rats. Toxicological findings from acute, subchronic, and chronic oral toxicity studies are consistently characterized by local stomach irritation, reduced food consumption, reduced body weight, and reduced weight gain. Misuse of these preparations may be deleterious to human health and when these chemicals are released through evaporation, they will have toxic and hazardous effects on the environment [41][42][43] . The most common disinfectant products on the market were formulated with these compounds and the formulations that we have examined showed antibacterial activity against E. coli and S. aureus. Formulations indicated in Table 1 as D6, D9, D11, D12 contain DDAC and the manufacturer reported on the label a description such as "ready to use product", without preliminary dilution. However, our data demonstrated the in vitro efficacy of these agents www.nature.com/scientificreports/ at very low concentrations, against Gram-positive and Gram-negative bacteria. In detail, the efficiency range was 0.001-0.0005 g/L against S. aureus and 0.0005 g/L against E. coli, respectively for D6 and D9. Whereas similar labeled products D11 and D12 exhibited a lower activity at the same concentration. The different activity was probably attributed to the different formulation compositions and the stability condition of disinfectant products. Therefore, our studies were focused on the comparison between commercial disinfectant formulations in real use.
Regarding the results obtained through the broth microdilution method, DDAC showed a similar trend compared to mixtures of benzalkonium chloride and DDAC, albeit with slightly greater efficacy. On the other hand, reported virucidal results showed a similar trend such as all solutions could be considered toxic against viruses to commercial concentration and "ready to use" solution, like D3 and D7, was very toxic against SARS-CoV-2 also at a concentration of 0,125 %. Alcohol based formulations were considered the best solutions for the surface disinfection process, even if in our investigation we obtained different results. In detail, for D1 formulation, ethanol more than 70%, recorded a MIC value lower than QACs compounds and an inhibition growth against S. aureus at 5 g/L and 45 g/L against E. coli; while isopropyl alcohol (D4) showed a bacterial growth inhibition up to 0.05 and 0.02 mg/L for S. aureus and E. coli, respectively. For the mixture of both alcohols, a MIC value greater than the individual component was detected, confirming the improved antibacterial efficacy of isopropyl alcohol.
In literature the optimum bactericidal concentration of alcohol solution reported is from 60 to 90%, although some studies showed that at a concentration beyond 70% the cell wall is sealed up preventing further entry of alcohol. For this reason, international organizations recommended formulations at 70% alcohol 44,45 . Many studies investigated the best type of alcohol and concentration for the disinfection of stethoscopes [46][47][48][49] . Stethoscopes are potential vectors for health care associated infections (HAI) and pose a potential risk in health care settings 50 .
The virucidal effects of alcohol were associated with their ability to disintegrate RNA, interfering with membrane integrity, and denaturation of viral proteins. Alcohols are amphoteric compounds and these properties promote the disintegration of the tertiary structure of proteins, causing the breakdown of the intramolecular hydrogen bonds within the structure. Chojnacki et al. 2021 evaluated the performance of 46 commercially hand sanitizers available on market for antibacterial activity toward prototypical Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial pathogens. Phenols and mixtures with alcohol disinfectants played an important role in hospital disinfectants thanks to their antimicrobial and virucidal efficacy 51 . Results confirmed the efficacy of phenol derivatives at a concentration of 0.5 to 5 g/L in a few minutes, as reported for HIV, through denaturing proteins and membrane disruption, which leads to leakage of components.

Conclusions
The experimental data highlighted the antimicrobial and virucidal activity of all commercial products evaluated on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and DNA and RNA viruses, such as HSV-1, and SARS-CoV-2. Additionally, our results demonstrated a better efficiency of QACs than others after dilution at 0,125%. The scientific evidence suggests an overuses of many commercial products inducing possible health effects, such as skin damage to the healthcare workers in their frequent daily use, and growing of environmental contamination. As noted, some disinfectant products appeared effective toward one or both organisms, whereas the antibacterial effects of other sanitizers seemed to wane. Further, there may be minor, yet appreciable, differences in the efficacy of QACs and alcohol-based formulations. Thus, it may be wise to implement formal requirements for efficacy data as a requisite for the continued production of disinfectants that have been introduced to the market under emergency COVID-19 authorization. Results of these studies indicate that antibacterial testing should probably be conducted and performed at lower concentrations to avoid excessive release of chemicals into the environment that can cause serious damage: e.g. modification of the environmental microbiota, potential threat to living beings and ecosystems, pollution of water and groundwater. Moreover, it will improve skin tolerance and consequently reduce occupational skin diseases such as irritant contact dermatitis and skin irritation processes, ensuring the health and safety protection of the operators at their workplace. These outcomes play a significant role in terms of user compliance with disinfection procedures, especially during the current COVID-19 pandemic. Similarly, it may be important to evaluate the effectiveness of sanitizers toward multiple strains of viruses.